Convergent synthesis of peptide nucleic acids by native chemical ligation

Article abstract of DOI:10.1021/ol051489+

(Chemical Equation Presented) A convergent strategy for synthesizing long contiguous PNA by a native chemical ligation-like technique of PNA segment couplings is presented. This approach required the synthesis of a new PNA-monomer featuring a 1-amino-2-th

Full text of DOI:10.1021/ol051489+

ORGANIC
LETTERS
2005
Vol. 7, No. 20
4365-4368
Convergent Synthesis of Peptide
Nucleic Acids by Native Chemical
Ligation
Christian Dose and Oliver Seitz*
Humboldt-UniVersita¨t zu Berlin, Institut fu¨r Chemie, Brook-Taylor-Strasse 2,
D-12489 Berlin, Germany
oliVer.seitz@chemie.hu-berlin.de
Received June 25, 2005
ABSTRACT
A convergent strategy for synthesizing long contiguous PNA by a native chemical ligation-like technique of PNA segment couplings is presented.
This approach required the synthesis of a new PNA-monomer featuring a 1-amino-2-thiol group. It is shown that the additional mercaptomethyl
group leaves the hybridization properties of PNA ligation products unaffected. Furthermore, rapid and efficient fluorescence labeling of the
ligation products is demonstrated.
Peptide nucleic acids (PNA) are achiral, uncharged DNA
analogues in which the nucleobases are attached to a
2-aminoethylglycine scaffold.1 The ability of PNA to bind
to complementary nucleic acids with very high affinity and
selectivity and the resistance to chemical and biological
degradation reactions has led to interesting applications in
DNA diagnostics,2 in antisense/antigene technology,3 and in
detection of RNA and DNA in cells.4 In particular, PNA-
oligomers spanning 18-25 nucleobases in length have been
used to monitor telomere dynamics,5 to inhibit human
telomerase,6 or to immobilize nucleic acids.7 PNA typically
is synthesized on the solid phase according to Fmoc or Boc
peptide synthesis protocols by iterative steps of protecting
group removal and coupling of Fmoc/Bhoc- or Boc/Cbz-
protected building blocks.8 However, the linear solid-phase
synthesis of PNAs longer than 20 nucleobases and/or of PNA
containing purine-rich sequences can result in low product
yield and/or low purity.9 In peptide chemistry, convergent
ligation techniques have been developed in order to overcome
the statistical accumulation of byproducts in the stepwise
synthesis of long peptides. The native chemical ligation
(NCL) reaction is a powerful method to join two unprotected
peptides in aqueous solution.10 In this reaction, a peptide
thioester is allowed to react with a cysteinyl peptide to first
form a thioester intermediate which, through a spontaneous
S f N acyl shift, furnishes the peptide bond in the ligation
product.11
(1) (a) Nielsen, P. E.; Berg, M. E. R.; Buchardt, O. Science 1991, 254,
1497. (b) Peptide Nucleic Acids: Protocols and Applications; Nielsen, P.
E., Egholm, M., Eds.; Horizon Scientific Press: Wymondham, U.K., 1999.
(2) (a) Ko¨hler, O.; Jarikote, D. V.; Seitz, O. ChemBioChem 2005, 5, 69.
(b) Ficht, S.; Mattes, A.; Seitz, O. J. Am. Chem. Soc. 2004, 126, 9970. (c)
Nielsen, P. E. Curr. Opin. Biotechnol. 2001, 12, 16.
(3) (a) Dean, D. A. AdV. Drug. DeliV. ReV. 2000, 44, 81. (b) Nielsen, P.
The native chemical ligation chemistry has also been used
to conjugate peptidic and nonpeptidic fragments to other
E. Curr. Med. Chem. 2001, 8, 545.
(4) Instructive examples include: (a) Oliveira K.; Procop, G. W.; Wilson,
D.; Coull, J.; Stender, H. J. Clin. Microbiol. 2002, 40, 247. (b) Rao, P. S.;
Tian, X.; Qin, W.; Aruva, M. R.; Sauter E. R.; Thakur, M. L.; Wickstrom,
E. Nuclear Med. Commun. 2003, 24, 857.
(5) Molenaar, C.; Wiesmeijer, K.; Verwoerd, N. P.; Khazen, S.; Eils,
R.; Tanke, H. J.; Dirks, R. W. EMBO J. 2003, 22, 6631.
(8) (a) Kova´cs, G.; Tima´r, Z.; Kupiha´r, Z.; Kele Z.; Kova´cs, L. J. Chem.
Soc., Perkin Trans. 1 2002, 1266. (b) Koch, T.; Hansen, H. F.; Andersen,
P.; Larsen, T.; Batz, H. G.; Otteson, K.; Ørum, H. J. Pept. Res. 1997, 49,
80.
(6) (a) Norton, J. C.; Piatyszek, M. A.; Wright, W. E.; Shay, J. W.; Corey,
D. R. Nature Biotech. 1996, 14, 615. (b) Herbert, B. S.; Pitts, A. E.; Baker,
S. I.; Hamilton S. E.; Wright, W. E.; Shay, J. W.; Corey, D. R. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 14276.
(9) Mayfield, L. D.; Corey, D. R. Anal. Biochem. 1999, 268, 401.
(10) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776.
(11) Wieland, T.; Bokelmann, E.; Bauer, L.; Lang, H. U.; Lau, H. Liebigs
Ann. Chem. 1953, 583, 129.
(7) Bentin T.; Nielsen P. E. Methods Mol. Biol. 2002, 208, 111.
10.1021/ol051489+ CCC: $30.25
© 2005 American Chemical Society
Published on Web 09/07/2005

biomolecules including DNA or PNA.12 In this work,
cysteine served as the ligation handle. However, the incor-
poration of cysteine in a convergent PNA-PNA fragment
coupling reaction would significantly distort the PNA
backbone and would hence reduce the affinity for target
nucleic acids. We hereby report the synthesis of a new
mercaptomethyl-modified PNA-monomer that allows the
application of native chemical ligation-like fragment coupling
reactions without detriment to backbone geometry. It is
shown that the DNA affinity of the PNA ligation products
is as high as the affinity of nonmodified PNA obtained by
linear solid-phase synthesis. Furthermore, rapid and efficient
fluorescence labeling of the ligation products is demonstrated.
The presented convergent approach enables the concise and
high-yielding synthesis of long fluorescently labeled PNA.
To enable PNA-fragment condensations by NCL-like
reactions a PNA-monomer is required that contains a
1-amino-2-thiol structure analogous to cysteine. We designed
the new PNA-monomer 1 as a structure analogue of a PNA
adenine monomer 2 (Figure 1). The introduction of amino
Scheme 1. Synthesis of Protected PNA Monomer 8 Suitable
for Native Chemical Ligation
yield. We next sought a method to couple 6-N-(benzyloxy-
carbonyl)-9-(carboxymethyl)adenine15 to the unreactive sec-
ondary amine in 6. Here, the use of pivaloyl chloride under
dry conditions proved superior to peptide couplings promoted
by carbodiimides, PyBOP, or HATU. The fully protected
mercaptomethyl adenine monomer 7 was obtained in 78%
yield. Finally, the methyl ester was saponified by treatment
with aqueous LiOH in THF, which completed a five-step
sequence and provided the optically active mercaptomethyl-
PNA monomer 8 in 52% overall yield.
The 1-amino-2-thiol-protected PNA-monomer 8 was next
employed in the solid-phase synthesis of PNA. Starting from
Fmoc-Gly-loaded MBHA-resin, Boc/Cbz-protected PNA
building blocks were assembled according to the Boc-strategy
(Scheme 2). After coupling of Boc/Trt/Cbz-protected adenine
building block 8 the Boc- and Trt-groups were removed by
adding TFA/m-cresol prior to treatment with TFMSA/TFA/
m-cresol required for Cbz and resin cleavage. Application
of this two-step protocol prevented the formation of tritylated
crude products observed in global deprotections with TFMSA/
TFA mixtures containing m-cresol, thioanisol, or triisopro-
pylsilane. The desired PNA conjugates 11, 12, and 13 were
obtained in 26%, 9%, and 4% overall yield, respectively,
after HPLC purification.
Figure 1. Structures of the PNA adenine monomer 2 and the
analogue 1 containing the 1-amino-2-thiol structure needed for
native chemical ligation-like reactions.
acid side chains such as from alanine, lysine, or arginine
into the PNA backbone has been reported.13 Surprisingly,
thiol groups have not been connected to the aminoethyl-
glycine scaffold. The envisaged application called for N-
terminal coupling of mercaptomethyl-modified PNA mono-
mer 1. It was therefore planned to employ acid-labile
protecting groups shown in Boc/Trt/Cbz-adenine monomer
8 which can be removed during the acid cleavage step of
Boc-based solid-phase PNA synthesis (Scheme 1). The
synthesis commenced from Boc/Trt-cysteine 3 which was
activated as a mixed anhydride by addition of isobutylchloro-
formate to enable the formation of the “Weinreb amide” 4.
Reduction with lithium aluminum hydride furnished the
corresponding aldehyde 5 in nearly quantitative overall
yield.14 This aldehyde was used in a reductive amination
reaction with glycine methyl ester hydrochloride, which
delivered the desired PNA-backbone building block 6 in 72%
The solid-phase synthesis of PNA-thioesters such as 14
presents a challenge.16 Most commonly, peptide thioesters
are prepared according to the Boc-strategy by using mer-
captopropionyl-based linkers.17 However, PNA-thioesters are
(14) Graham, S. L.; deSolms, S. J.; Giuliani E. A.; Kohl, N. E.; Mosser,
S. D.; Oliff, A. I.; Pompliano D. L.; Rands E.; Breslin, M. J.; Deana, A.
A.; Garsky, V. M.; Scholz, T. H.; Gibbs, J. B.; Smith, R. L. J. Med. Chem.
1994, 37, 725.
(15) Thomson, S. A.; Josey, J. A.; Cadilla, R.; Gaul, M. D.; Hassman,
C. F.; Luzzio, M. J.; Pipe, A. J.; Reed, K. L.; Ricca, D. J.; Wiethe, R. W.;
Noble, S. A. Tetrahedron 1995, 51, 6179.
(16) de Koning, M. C.; Filippov, V. D.; van der Marel, G. A.; van Boom,
J. H.; Overhand, M. Tetrahedron Lett. 2003, 44, 7597.
(17) Hackeng, T. M.; Griffin J. H.; Dawson. P. E. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 10068.
(12) (a) Stetsenko D. A.; Gait, M. J. J. Org. Chem. 2000, 65, 4900. (b)
de Koning, M. C.; van der Knaap, M.; Petersen, L.; van den Elst, H.; van
der Marel, G. A.; Overhand, M.; Filippov, D. V. Synlett 2005, 4, 595. (c)
de Koning M. C.; Filippov D. V.; van der Marel G. A.; van Boom J. H.;
Overhand M. Eur. J. Org. Chem. 2004, 4, 850.
(13) (a) Kosynkina, L.; Wang, W.; Liang, T. C. Tetrahedron Lett. 1994,
35, 5173. (b) Pu¨schl, A.; Sforza, S.; Haaima, G.; Dahl, O.; Nielsen, P. E.
Tetrahedron Lett. 1998, 39, 4707.
4366
Org. Lett., Vol. 7, No. 20, 2005

Table 1. PNA Conjugates, Ligation Products, and Yields of the Ligation Reactionsa
nucleophile
electrophile
ligation product
yieldb/%
ActtccccacS(CH2)2COGlyCONH
ActtccccacS(CH2)2COGlyCONH
14
14
15
Acttccccac(HSCH2)acctccaGlyCONH
16
17
18
72
67
69
2
2
2
11
12
13
Acttccccac(HSCH2)atccttgatcttttcGlyCONH
Acccctaaccct(HSCH2)aaccctaaccctaaGlyCONH
2
AcccctaaccctS(CH2)2COGlyCONH
2
2
a Reaction conditions: 0.1 mM reactants in a buffer containing 10 mM tris(2-carboxyethyl)phosphine hydrochloride, 10 mM MESNA, and 100 mM
NaH2PO4 adjusted to pH 7.4. b Product yields after 14 h determined by HPLC analysis.
subject to cyclization reactions which furnish six-membered
lactams. Indeed, the linear solid-phase synthesis of PNA-
thioester 14 proceeded with low yield due to the formation
of C f N-terminally truncated PNA byproducts (Figure S1,
Supporting Information). The formation of these so-called
inverted truncation products can be explained by assuming
that the reactive thiol group obtained after cyclization
experienced repeated cycles of acylation and lactamization.
Presumably, cyclative PNA-cleavage predominantly occurred
during coupling of the second monomer. To reduce the
amount of the undesired lactamization reaction, the usual
coupling procedure (0.1 M PNA building block, 0.15 M
N-methylmorpholine, 0.1 M PyBOP) was changed to less
basic conditions. The second PNA building block was
coupled at 0.125 M concentration by performing the in situ
neutralization with 1.1 equiv (based on acyl donor) of 2,6-
lutidine (pKa 6.65) instead of 1.5 equiv of N-methylmor-
pholine (pKa 7.38). After the critical extension of resin-bound
PNA-monomer, the subsequent coupling reactions proceeded
as previously described. Ultimately, acidolytic release pro-
vided PNA-thioesters 14 and 15 as the main products in 11%
and 3% yield after HPLC purification.
corresponded to ligation product 16 (Figure 2). After 14 h
reaction time PNA ligation product 16 was formed in 72%
yield.
Figure 2. HPLC of ligation between PNA-thioester 14 and PNA-
thiol conjugate 11. Reaction conditions: see Table 1.
The feasibility of the synthesis of large PNA was
investigated by allowing PNA thioester 14 to react with
16mer PNA 12 (Table 1). The ligation proceeded smoothly
and provided 24mer PNA in 67% yield after 14 h reaction
time. Furthermore, the ligation of 10mer PNA-thioester 15
with 14mer PNA 13 was explored. The sequence of PNA
18 corresponds to a tetrad of the C3TA2 repeat that should
allow the probing of telomer length in human cells.18 The
ligation of 15 with 13 succeeded just as well as the previous
ones supplying product 18 in 69% yield. These results
provided clear evidence that the synthesized PNA-monomer
8 enables fragment coupling of PNA-oligomers by an
efficient NCL-like reaction.
Scheme 2. Solid Phase Synthesis.
There are numerous applications in DNA diagnostics and
molecular biology which rely on the introduction of fluoro-
phores to PNA and DNA. In addition to its usefulness as
ligation handle in PNA nucleophiles 11, 12, and 13, the
mercaptomethyl groups in PNA ligation products 16, 17, and
18 also offer conjugation sites for the selective reaction with
soft electrophiles. Ligation product 16 was allowed to react
with the thiol reactive dye 7-diethylamino-3-(4′-maleimidyl
phenyl)-4-methylcoumarin (CPM) (Figure 3). Near-quantita-
tive labeling of PNA 16 was accomplished by performing
The reactivity of PNA-thioester 14 was studied in explor-
ing the native chemical ligation-like reaction with 1-amino-
2-thiol PNA 11. The reaction was carried out in the presence
of 10 mM tris(2-carboxyethyl)phosphine hydrochloride to
maintain a reducing environment. Sodium 2-mercaptoethane-
sulfonate (MESNA) was added for in situ generation of a
more reactive thioester 19. HPLC analysis of aliquots showed
a new peak that appeared at 15.6 min with a mass that
(18) Landsdorp, P. M.; Verwoerd, N. P.; van de Rijke, F. M.; Dragowska,
V.; Little, M.-T.; Dirks, R. W.; Raap, A. K.; Tanke, H. J. Hum. Mol. Genet.
1996, 5, 685.
Org. Lett., Vol. 7, No. 20, 2005
4367

Table 2. Melting Temperatures of DNA·PNA Duplexesa
15 mer PNA
24 mer PNA
(HSCH2)PNA PNA CPM-PNA (HSCH2)PNA PNA
PNA•DNA
TM/°C
16•23
71
21•23
70
20•23
69
17•24
76
22•24
75
a DNA-oligomers: 5′TGGAGGTGTGGGGAA3′ 23 and 5′GAAAAGAT-
CAAAGGATGTGGGGA3′ 24. Measurements were performed in a buffer
containing 1 µM DNA/PNA oligomers, 10 mM NaCl, and 10 mM NaH2PO4
at pH 7.0. Denaturation: 15-90 °C, 1 °C/min, monitored at 260 nm.
and 17‚24 had no negative effect on the DNA affinity of
PNA probes. Likewise, the presence of the CPM fluorophore
in CPM-PNA‚DNA duplex 20‚23 (TM ) 69 °C) led to a
minor decrease of duplex stability. The results from CPM
labeling and hybridization studies suggest that the mercap-
tomethyl group in PNAs 16, 17, and 18 are directed to the
exterior of the PNA‚DNA duplex where unhindered access
for the attachment of reporter groups is granted.
In conclusion, we have introduced convergent access to
long PNA by developing a native chemical ligation-like
technique of PNA segment couplings. The required PNA-
monomer features a 1-amino-2-thiol function and was
synthesized in five steps starting from commercially available
starting materials. The additional mercaptomethyl group in
PNA ligation products such as 16 has no effect on DNA
affinity. In addition to its utility in PNA fragment ligations
the mercaptomethyl group also provides a selectively ad-
dressable conjugation site. The presented convergent ap-
proach may find applications in the synthesis and fluores-
cence labeling of difficult PNA-oligomers (>20 bases, purine
rich, repetitive nucleotides) and in DNA-template-controlled
ligation reactions.
Figure 3. HPLC analysis of thiol specific labeling of PNA
conjugate 16 with 7-diethylamino-3-(4′-maleimidylphenyl)-4-me-
thylcoumarine (CPM). Reaction conditions: 0.1 mM of 16 in a
buffer containing 100 mM NaH2PO4 and 1 mM CPM adjusted to
pH 7.4.
the alkylation at 0.1 mM ligation product 16 and 10-fold
excess of CPM. The reaction went to completion after only
1 min reaction time, illustrating the high reactivity of the
mercaptomethyl group. HPLC analysis showed that product
20 at 23.2 min (m/z ) 4451) exists as mixture of diastereo-
mers due to the use of optically active PNA-monomer 8.
Long PNA is required in intracellular applications such
as in fluorescence in situ hybridization (FISH) in which the
stability of probe-target complexes must be high.19 The
influence of the mercaptomethyl and CPM modifications was
investigated by comparing the thermal stability of PNA‚DNA
duplexes containing modified PNAs 16, 17, and 20 with
duplexes containing nonmodified PNA Acttccccacacctcca-
GlyCONH2 21 and ActtccccacatcctttgatcttttcGlyCONH2 22 (Table
2). Note that the linear solid-phase synthesis of 24mer PNA
22 was cumbersome and required 13 double couplings. The
melting analyses revealed melting temperatures TM of 71 and
76 °C for mercaptomethyl-PNA‚DNA duplexes 16‚23 and
17‚24, respectively. A comparison with the TM of 70 and 75
°C determined for the corresponding nonmodified PNA‚DNA
duplexes 21‚23 and 22‚24, respectively, indicated that the
introduction of mercaptomethyl groups in duplexes 16‚23
Acknowledgment. We acknowledge support from DFG,
Fonds der Chemischen Industrie, and Schering AG.
Supporting Information Available: Experimental pro-
cedures and characterization data for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(19) Baerlocher, G. M.; Mak, J.; Tien, T.; Landsorp, P. M. Cytometry
2002, 47, 89.
OL051489+
4368
Org. Lett., Vol. 7, No. 20, 2005